The proposal outlines a 5 year training plan to facilitate Dr. Einav's career development as an independent academic physician-scientist. Dr. Einav recently completed clinical training in infectious diseases at Stanford University. The proposed project takes advantage of outstanding technical and intellectual resources available at Stanford for the study of hepatitis C virus (HCV). Drs. Glenn and Quake who will serve as co-mentors for the project are both internationally recognized authorities on the molecular virology of HCV, and microfluidics, respectively. Current therapies are inadequate for most of the 170 million people infected with HCV worldwide. Our long-term objectives are to better understand the molecular virology of HCV and translate this knowledge into new antiviral strategies. This project's goal is to better understand non-structural 4B's (NS4B) role in HCV RNA replication and cellular transformation and enable the design of new anti-HCV strategies. Our overall hypothesis is that two different heretofore unrecognized functions of NS4B; nucleotide binding and hydrolysis and HCV RNA binding, are each essential for mediating NS4B's role in HCV replication, and the former is also essential for mediating NS4B's role in cellular transformation. Thus approaches designed to disrupt these functions may be potentially used to inhibit HCV replication. Pharmacologic inhibition of the nucleotide binding motif (NBM) may also inhibit the associated hepatoma. Our preliminary results suggest that NS4B binds and hydrolyzes GTP and binds HCV RNA. Genetically disrupting the conserved NBM and RNA binding domain impairs their biochemical activity and dramatically inhibits HCV replication. Furthermore, NS4B can mediate cellular transformation and tumor formation in mice independently of the Ha-ras gene. Specific aims and research design: 1) To characterize RNA binding by NS4B and its NBM by defining additional critical elements that mediate these functions and by determining the substrate specificity of the RNA binding. Biochemical analysis of RNA binding will be done using a novel microfluidics platform. 2) To define NS4B's role in HCV RNA replication by determining the genetic effects of disruption of the various elements of the NBM and RNA binding elements. 3) To define NS4B's role in malignant transformation by determining the role of the NBM in mediating this activity, by determining the role of NS4B in causing hepatoma in humans using sequence analysis, and by characterizing gene expression profiling in NS4B transformed clones. Taken together, more effective therapies are urgently needed against HCV, a major cause of viral hepatitis. We hope that this study will help translate our molecular virology findings into the development of new classes of anti-HCV agents. The latter could not only complement current drug cocktails, but by attacking novel targets would hopefully increase the therapeutic efficacy of the resulting combination.